Methods, devices, and systems using acoustic ejection for depositing fluid droplets on a sample surface for analysis

ABSTRACT

Provided is a method for preparing a sample surface for analysis that involves placing a sample surface in droplet-receiving relationship to a reservoir containing an analysis-enhancing fluid. Typically, the analysis-enhancing fluid is comprised of a mass spectrometry matrix material and a carrier fluid, and the carrier fluid is comprised of a low volatility solvent. A droplet of the analysis-enhancing fluid from the reservoir such that the droplet is deposited on the sample surface at a designated site. Such ejection is typically, but not necessarily carried out through the application of focused acoustic energy. Then, the sample is subjected to conditions sufficient to allow the analysis-enhancing fluid to interact with the sample surface to render the sample surface suitable for analysis. Optionally, the sample is analyzed at the selected site. Also provided are systems and devices for preparing a sample surface for analysis.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a continuation-in-part of U.S. patent application Ser. No.10/157,755, filed May 28, 2002 now U.S. Pat. No. 6,707,038, which is acontinuation-in-part of U.S. patent application Ser. No. 10/087,372,filed Mar. 1, 2002, which is a continuation-in-part of U.S. patentapplication Ser. No. 10/066,546, filed Jan. 30, 2002 now U.S. Pat. No.6,710,335, which is a continuation-in-part of U.S. patent applicationSer. No. 09/784,705, filed Feb. 14, 2001 now U.S. Pat. No. 6,603,118,the disclosures of which are incorporated by reference herein.

TECHNICAL FIELD

This invention relates generally to methods, devices, and systems fordepositing fluids on a surface of a sample. More particularly, theinvention relates to the use of nozzleless acoustic ejection to depositdroplets of analysis-enhancing fluid on designated sites of a samplesurface. The invention is especially useful in mass spectrometricimaging of tissue surfaces and for facilitating the compositionalanalysis of biological samples.

BACKGROUND

Mass spectrometry is a well-established analytical technique in whichsample molecules are ionized and the resulting ions are sorted bymass-to-charge ratio. Advances in mass spectrometry have made itpossible to obtain detailed information regarding a wide variety ofsample surface types. In the semiconductor industry, for example,secondary ion mass spectrometry is used to determine the composition ofmicroscopic regions of wafer surfaces. As another example, in thebiotechnology arena, surface-based mass spectrometry is used to analyzesingle nucleotide polymorphisms in microarray formats. See, e.g., U.S.Pat. No. 6,322,970 to Little et al.

Matrix-Assisted Laser Desorption Ionization (MALDI) is an ionizationtechnique commonly used for mass spectrometric analysis of large and/orlabile biomolecules, such as nucleotidic and peptidic oligomers,polymers, and dendrimers, as well as for analysis of non-biomolecularcompounds, such as fullerenes. MALDI is considered a “soft” ionizingtechnique in which both positive and negative ions are produced. Thetechnique involves depositing a small volume of sample fluid containingan analyte on a substrate comprised of a photon-absorbing matrixmaterial selected to enhanced desorption performance. See Karas et al.(1988), “Laser Desorption Ionization of Proteins with Molecular MassesExceeding 10,000 Daltons,” Anal. Chem. 60:2299-2301. The matrix materialis usually a crystalline organic acid that absorbs electromagneticradiation near the wavelength of the laser. When co-crystallized withanalyte, the matrix material assists in the ionization and desorption ofanalyte moieties. The sample fluid typically contains a solvent and theanalyte. Once the solvent has been evaporated from the substrate, theanalyte remains on the substrate at the location where the sample fluidis deposited. Photons from a laser strike the substrate at the locationof the analyte and, as a result, ions and neutral molecules are desorbedfrom the substrate. MALDI techniques are particularly useful inproviding a means for efficiently analyzing a large number of samples.In addition, MALDI is especially useful in the analysis of minuteamounts of sample that are provided over a small area of a substratesurface.

Surface Enhanced Laser Desorption Ionization (SELDI) is another exampleof a surface-based ionization technique that allows for high-throughputmass spectrometry. SELDI uses affinity-capture reagents, such asantibodies, to collect samples from a complex mixture, which allows insitu purification of the analyte followed by conventional MALDIanalysis. Typically, SELDI is used to analyze complex mixtures ofproteins and other biomolecules. SELDI employs a chemically reactivesurface such as a “protein chip” to interact with analytes, e.g.,proteins, in solution. Such a surface selectively interacts withanalytes and immobilizes them thereon. Thus, analytes can be partiallypurified on the chip and then quickly analyzed in the mass spectrometer.By providing different reactive moieties at different sites on asubstrate surface, throughput may be increased.

Recently, mass spectrometry techniques involving laser desorption havebeen adapted for cellular analysis. Cellular assays such as massspectrometry are carried out to provide critical information for theunderstanding of complex cell functions. U.S. Pat. No. 5,808,300 toCaprioli, for example, describes a method for imaging biological sampleswith mass spectrometry using surface-based ionization. This methodallows users to measure the distribution of a specific element or smallmolecule within biological specimens such as tissue slices or individualcells. In particular, the method can be used for the analysis ofspecific peptides in whole cells, e.g., by obtaining signals forpeptides and proteins directly from tissues and blots of tissues. Inaddition, the method has been used to desorb relatively large proteinsfrom tissues and blots of tissues in the molecular weight range beyondabout 80 kilodaltons. From such samples, hundreds of peptide and proteinpeaks can be recorded in the mass spectrum produced from a singlelaser-ablated site on the sample. When a laser ablates the surface of asample at multiple sites and the mass spectrum from each site is savedseparately, a data array is produced, which contains the relativeintensity of any given mass at each site. An image of the sample surfacecan then be constructed for any given molecular weight, effectivelyrepresenting a compositional map of the sample surface.

One important issue to successful MALDI and MALDI-like profiling andimaging as described above is the controlled application of amass-spectrometry matrix material to the tissue surface, either as aseries of features or as a continuous coating so as to provide massspectrometry matrix material at each site of laser ablation. Forexample, as described in U.S. Pat. No. 5,808,300 to Caprioli, the massspectrometry matrix material may be applied as a continuous and uniformcoating of less than about 50 micrometers in thickness. In order toapply the mass spectrometry matrix material in a controlled manner,carefully metered amounts of sample fluids should be accurately andprecisely placed on a sample surface. The ability to closely comparerelative abundances of a given protein between two tissues is dependenton the application of matrix in exactly the same way to both tissues.

Most current small-volume dispensing techniques, however, are notsuitable for precise and reproducible matrix material application, dueto limitations in volume or in accuracy of placement. For example,capillaries having a small interior channel (e.g., Eppendorf-typecapillaries) are often used to transfer fluids from a pool of fluid.Their tips are submerged in the pool in order to draw fluid therefrom.In order to provide sufficient mechanical strength for handling,however, such capillaries must have a large wall thickness as comparedto the interior channel diameter. Thus, the physical dimensions of suchcapillaries limit their fluid-handling capability. In addition, sinceany wetting of the exterior capillary surface results in fluid waste,the high ratio of wall thickness to channel diameter exacerbates fluidwaste. Also, the pool has a minimum required volume determined not bythe fluid introduced into the capillary but, rather, by the need toimmerse the large exterior dimension of the capillary. As a result, thefluid volume required for capillary submersion may be more than an orderof magnitude larger than the fluid volume transferred into thecapillary.

A number of patents have described the use of acoustic energy inprinting. For example, U.S. Pat. No. 4,308,547 to Lovelady et al.describes a liquid drop emitter that utilizes acoustic principles inejecting droplets from a body of liquid ink onto a moving document toform characters or bar codes thereon. As described in a number of U.S.patent applications, acoustic ejection provides for highly accuratedeposition of minute volumes of fluids on a surface, wherein dropletvolume—and thus “spot” size on the substrate surface—can be carefullycontrolled, and droplets can be precisely directed to particular siteson a substrate surface. See, e.g., U.S. Patent Application PublicationNo. 2002037579 to Ellson et al. In other words, nozzleless fluiddelivery provides high fluid-delivery efficiency through accurate andprecise droplet placement. Nozzleless fluid ejection also provides ahigh level of control over ejected droplet size.

Acoustic ejection is a technique that is well suited for depositingminute volumes of fluids on a surface because the technique allows forcontrol over droplet volume and thus “spot” size on the surface, as wellas control over the trajectory of ejected droplets and the preciselocation of the deposition sites on the surface. See, e.g., U.S. PatentApplication Publication No. 20020037579 to Ellson et al. Whilenozzleless fluid ejection has generally been appreciated for inkprinting applications, acoustic deposition is a generally unknowntechnique in the field of cellular analysis. Recently, focused acousticenergy has been used to manipulate cells and engage in cell sorting. SeeU.S. Patent Application Publication Nos. 20020064808, 20020064809,20020090720, and 20020094582 to Mutz et al. In addition, as cellularassays often involve the immobilization of sample cells on a substratesurface and the controlled exposure of the cells to one or more fluids,there exist opportunities to improve cellular assay and analysistechniques through the use of acoustic ejection, particularly when suchassays require the precise and accurate handling of small volumes offluid. For example, U.S. Patent Application Publication No. 20020171037to Ellson et al. describes the use of acoustic ejection for preparingand analyzing a cellular sample surface. Nozzleless acoustic ejection isused to deposit mass spectrometry matrix material at designated sites ona sample surface to form either a uniform matrix material layer or anarray of individual sites. In addition, U.S. Patent ApplicationPublication No. 20020195538 to Ellson et al. describes the use ofacoustic ejection to selectively deposit analysis-enhancing fluidaccording to the surface characteristics of the cellular samples.

As alluded to above, conventional analysis-enhancing fluids for use inmass spectrometry are typically comprised of a mass spectrometry matrixmaterial dissolved in a volatile carrier fluid. Once deposited on asample surface, the carrier fluid is evaporated, thereby allowing thematrix material to precipitate and crystallize with the sample. It hasrecently been discovered, however, that such conventionalanalysis-enhancing fluids are not optimal for use in mass spectrometrywhen dispensed as low-volume droplets under ordinary dispensingconditions, because such fluids do not allow the matrix material toproperly crystallize with the sample.

Accordingly, there is a need for methods and systems that overcome thedisadvantages and limitations associated with previously knowntechnologies.

SUMMARY OF THE INVENTION

Accordingly, one embodiment of the invention relates to method forpreparing a sample surface for analysis. In general, a reservoircontaining an analysis-enhancing fluid is provided, and a surface of asample is placed in droplet-receiving relationship to thefluid-containing reservoir. In some instances, the reservoir has avolume no greater than 100 nL, and the sample is a cellular sample. Adroplet of the analysis-enhancing fluid from the reservoir such that thedroplet is deposited on the sample surface at a designated site. Suchejection is typically, but not necessarily carried out through theapplication of focused acoustic energy. Then, the sample is subjected toconditions sufficient to allow the analysis-enhancing fluid to interactwith the sample surface to render the sample surface suitable foranalysis. Optionally, the sample is analyzed at the selected site.

Typically, the analysis-enhancing fluid is comprised of ananalysis-enhancing moiety and a carrier fluid. In some instances, thecarrier fluid is comprised of a low volatility solvent that has aboiling point greater than 100° C. at a pressure of 1 atmosphere. Inaddition, the carrier fluid may further comprise a high volatilitysolvent that has a boiling point less than 100° C. at a pressure of 1atmosphere. The analysis-enhancing moiety may comprise a massspectrometry matrix material. In such a case, energy is applied to thedesignated site in a manner effective to release sample molecules fromthe sample surface for analysis, e.g., MALDI-TOF mass spectrometricanalysis.

The invention is particularly suited for instances in which the ejecteddroplet has a small volume to as to control the rate of solventevaporation. Typically, the volume of the ejected droplet is no greaterthan about 50 pL. Preferably, the volume of the ejected droplet is nogreater than about 10 pL More preferably, the volume of the ejecteddroplet is no greater than about 1 pL. Optimally, the volume of theejected droplet is less than about 1 pL, e.g., in the range of about0.025 pL to about 1 pL.

There are a number of ways to subject the sample to conditionssufficient to allow the analysis-enhancing fluid to interact with thesample surface to render the sample surface suitable for analysis. Whenthe analysis-enhancing fluid is comprised of a low volatility solvent,the sample may be subjected to a temperature greater than 25° C. and/orto a pressure lower than about 1 atmosphere. When the analysis-enhancingfluid is comprised of a high volatility solvent, the sample may besubjected to a temperature lower than 25° C. and/or a pressure greaterthan 1 atmosphere. In some instances, the sample to an atmosphere thatis at least about 30% saturated with the carrier fluid. In addition orin the alternative, the sample is subjected to conditions such that thecarrier fluid exhibits a volatility quotient of at least about 30.

The invention also provides a system for depositing ananalysis-enhancing fluid on a surface of a sample. The system iscomprised of a reservoir containing an analysis-enhancing fluid asdescribed above, an acoustic ejector comprising an acoustic radiationgenerator for generating acoustic radiation and a focusing means forfocusing the acoustic radiation generated, a means for positioning theacoustic ejector in acoustic coupling relationship to the reservoir; anda means for positioning the sample such that a designated site on thesample surface is positioned in droplet-receiving relationship to thereservoir. Optionally, the system may further comprise a means, e.g., alaser, for applying energy to the designated site to effect release andionization of sample molecules from the sample surface for analysis, andan analyzer, e.g., a mass spectrometer, positioned to receive ionizedsample molecules released from the sample surface.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is described in detail below with reference to thefollowing drawings, wherein like reference numerals indicate acorresponding structure throughout the several views. The figuresillustrate aspects of the invention that involve the analysis of atissue sample through laser desorption-based mass spectrometry.

FIG. 1 schematically illustrates the deposition of a firstanalysis-enhancing fluid on a surface of a tissue sample at a firstsite.

FIG. 2 schematically illustrates the deposition of a secondanalysis-enhancing fluid on a surface of the tissue sample at a secondsite.

FIG. 3 schematically illustrates the ionization of the tissue samplewhose preparation is depicted in FIGS. 1 and 2.

FIG. 4 schematically illustrates in top view a tissue sample on asubstrate, wherein the tissue sample exhibits a nonuniform exposedsurface.

FIG. 5 schematically illustrates in side view the imaging of the exposedsurface of the tissue sample depicted in FIG. 4.

FIG. 6 schematically illustrates in side view the deposition of a firstanalysis-enhancing fluid on a surface of the tissue sample depicted inFIG. 4 at a first site selected according to a first surfacecharacteristic.

FIG. 7 schematically illustrates in side view the deposition of a secondanalysis-enhancing fluid on a surface of the tissue sample depicted inFIG. 4 at a second site selected according to a second surfacecharacteristic.

FIG. 8 schematically illustrates in side view the ionization of thetissue sample whose preparation is depicted in FIGS. 6 and 7.

FIG. 9 schematically illustrates in top view a tissue sample exhibitinga nonuniform upper surface having droplets of differentanalysis-enhancing fluids deposited thereon at different sites. Thesesites are selected according to specific surface characteristics, andthe sites associated with each analysis-enhancing fluid form an array.

DETAILED DESCRIPTION OF THE INVENTION

Definitions and Overview:

Before describing the present invention in detail, it is to beunderstood that this invention is not limited to specific fluids,biomolecules, or device structures, as such may vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting.

It must be noted that, as used in this specification and the appendedclaims, the singular forms “a,” “an,” and “the” include both singularand plural referents unless the context clearly dictates otherwise.Thus, for example, reference to “a reservoir” includes a plurality ofreservoirs as well as a single reservoir, reference to “a fluid”includes a mixture of fluids as well as a single fluid, reference to “abiomolecule” includes a combination of biomolecules as well as a singlebiomolecule, reference to “a characteristic” includes a plurality ofcharacteristics as well as a single characteristic, and the like.

In describing and claiming the present invention, the followingterminology will be used in accordance with the definitions set outbelow.

The terms “acoustic coupling” and “acoustically coupled” as used hereinrefer to a state wherein an object is placed in direct or indirectcontact with another object so as to allow acoustic radiation to betransferred between the objects without substantial loss of acousticenergy. When two items are indirectly acoustically coupled, an “acousticcoupling medium” is needed to provide an intermediary through whichacoustic radiation may be transmitted. Thus, an ejector may beacoustically coupled to a fluid, e.g., by immersing the ejector in thefluid or by interposing an acoustic coupling medium between the ejectorand the fluid to transfer acoustic radiation generated by the ejectorthrough the acoustic coupling medium and into the fluid.

The terms “acoustic radiation” and “acoustic energy” are usedinterchangeably herein and refer to the emission and propagation ofenergy in the form of sound waves. As with other waveforms, acousticradiation may be focused using a focusing means, as discussed below.

The term “adsorb” as used herein refers to the noncovalent retention ofa molecule or a cell by a surface. That is, adsorption occurs as aresult of noncovalent interaction between a surface and adsorbingmoieties present on the molecule. Adsorption may occur through hydrogenbonding, van der Waal's forces, polar attraction, or electrostaticforces (i.e., through ionic bonding). Examples of adsorbing moietiesinclude, but are not limited to, amine groups, carboxylic acid moieties,hydroxyl groups, nitroso groups, sulfones, and the like.

The term “adsorb” is often used in the context of substrate or samplesurfaces. The substrate or sample surface commonly may be functionalizedwith adsorbent moieties to interact in a certain manner, as when thesurface is functionalized with amino groups to render it positivelycharged in a pH-neutral aqueous environment. Likewise, adsorbatemoieties may be added in some cases to effect adsorption, as when abasic protein is fused with an acidic peptide sequence to renderadsorbate moieties that can interact electrostatically with a positivelycharged adsorbent moiety.

The term “array” as used herein refers to a two-dimensional arrangementof features, such as an arrangement of reservoirs (e.g., wells in a wellplate) or an arrangement of fluid droplets or molecular moieties on asubstrate surface (as in an oligonucleotide or peptide array). Arraysare generally comprised of features regularly ordered in, for example, arectilinear grid, parallel stripes, spirals, and the like, butnon-ordered arrays may be advantageously used as well. An array differsfrom a pattern in that patterns do not necessarily contain regular andordered features. In addition, arrays and patterns formed by thedeposition of ejected droplets on a surface, as provided herein, areusually substantially invisible to the unaided human eye. Arraystypically, but do not necessarily, comprise at least about 4 to about10,000,000 features, generally in the range of about 4 to about1,000,000 features.

The term “attached,” as in, for example, a substrate surface having amolecular moiety “attached” thereto, includes covalent binding,adsorption, and physical immobilization. The terms “binding” and “bound”as used herein are identical in meaning to the term “attached.”

The terms “biomolecule” and “biological molecule” are usedinterchangeably herein to refer to any organic molecule—whethernaturally occurring, recombinantly produced, or chemically synthesizedin whole or in part—that is, was, or can be a part of a living organism.The terms encompass, for example, nucleotides, amino acids, andmonosaccharides, as well as oligomeric and polymeric species such asoligonucleotides and polynucleotides; peptidic molecules such asoligopeptides, polypeptides, and proteins; polysaccharides such asdisaccharides, oligosaccharides, mucopolysaccharides, and peptidoglycans(peptidopolysaccharides); and the like. The terms also encompassribosomes, enzyme cofactors, pharmacologically active agents, and thelike.

It will be appreciated that, as used herein, the terms “nucleoside” and“nucleotide” refer to nucleosides and nucleotides containing not onlythe conventional purine and pyrimidine bases, i.e., adenine (A), thymine(T), cytosine (C), guanine (G), and uracil (U), but also protected formsthereof, e.g., wherein the base is protected with a protecting groupsuch as acetyl, difluoroacetyl, trifluoroacetyl, isobutyryl, or benzoyl,and purine and pyrimidine analogs. Suitable analogs will be known tothose skilled in the art and are described in the pertinent texts andliterature. The terms “peptide,” “peptidyl,” and “peptidic” as usedthroughout the specification and claims are intended to include anystructure comprised of two or more amino acids. For the most part, thepeptides in the present arrays comprise about 5 to 10,000 amino acids,preferably about 5 to 1,000 amino acids. The amino acids forming all ora part of a peptide may be any of the twenty conventional, naturallyoccurring amino acids, i.e., alanine (A), cysteine (C), aspartic acid(D), glutamic acid (E), phenylalanine (F), glycine (G), histidine (H),isoleucine (I), lysine (K), leucine (L), methionine (M), asparagine (N),proline (P), glutamine (Q), arginine (R), serine (S), threonine (T),valine (V), tryptophan (W), and tyrosine (Y). Any of the amino acids inthe peptidic molecules forming the present arrays may be replaced by anonconventional amino acid. Additional information relating to the terms“biomolecule,” “nucleotide,” and “peptide” can be found in U.S. PatentApplication Publication No. 20020037579 to Ellson et al.

The terms “cell” and “cellular” are used herein with their ordinarybiological meaning and refer to the smallest structural unit of anorganism, living or not, that is capable of independent functioning, andincludes one or more of nuclei, cytoplasm, and various organelles, allsurrounded by a semipermeable membrane. The term “cellular sample” asused herein refers to a sample that typically contains a plurality ofcells. The cells may be of the same type or different types. When thecellular sample contains a single cell, the cell is large, e.g., afertilized or unfertilized cell. Any number of processing techniques maybe used to prepare a cellular sample. For example, the cells may becultured or extracted from living or dead organisms. In addition, thecellular samples may be prepared for microscopy, e.g., through smears,squashes, mounts, and sections.

The term “fluid” as used herein refers to matter that is nonsolid or atleast partially gaseous and/or liquid. A fluid may contain a solid thatis minimally, partially, or fully solvated, dispersed, or suspended.Examples of fluids include, without limitation, aqueous liquids(including water per se and salt water) and nonaqueous liquids such asorganic solvents and the like. As used herein, the term “fluid” is notsynonymous with the term “ink” in that an ink must contain a colorantand may not be gaseous. However, the term “fluid” is intended toencompass fluid inks.

Thus, the term “analysis-enhancing fluid” refers to any fluid that maybe required or desired for use in conjunction with an analyticaltechnique. Typically, analysis-enhancing fluids are employed to increasethe yield of useful information during surface analysis.

The terms “focusing means” and “acoustic focusing means” refer to ameans for causing acoustic waves to converge at a focal point by eithera device separate from the acoustic energy source that acts like anoptical lens, or by the spatial arrangement of acoustic energy sourcesto effect convergence of acoustic energy at a focal point byconstructive and destructive interference. A focusing means may be assimple as a solid member having a curved surface, or it may includecomplex structures such as those found in Fresnel lenses, which employdiffraction in order to direct acoustic radiation. Suitable focusingmeans also include phased array methods as known in the art and asdescribed, for example, in U.S. Pat. No. 5,798,779 to Nakayasu et al.and Amemiya et al. (1997) Proceedings of the 1997 IS&TNIP13International Conference on Digital Printing Technologies, pp. 698-702.

The term “ion” is used in its conventional sense to refer to a chargedatom or molecule, i.e., an atom or molecule that contains an unequalnumber of protons and electrons. Positive ions contain more protons thanelectrons, and negative ions contain more electrons than protons.Ordinarily, an ion of the present invention is singly charged, but mayin certain instances have a multiple charge.

Accordingly, the term “ionization chamber” as used herein refers to achamber in which ions are formed from samples—fluid or otherwise—thatcontain a sample molecule.

The term “moiety” as used herein refers to any particular composition ofmatter, e.g., a molecular fragment, an intact molecule (including amonomeric molecule, an oligomeric molecule, or a polymer), or a mixtureof materials (for example, an alloy or a laminate).

The term “near” as used herein refers to the distance from the focalpoint of the focused acoustic radiation to the surface of the fluid fromwhich a droplet is to be ejected and indicates that the distance shouldbe such that the focused acoustic radiation directed into the fluidresults in droplet ejection from the fluid surface so that one ofordinary skill in the art will be able to select an appropriate distancefor any given fluid using straightforward and routine experimentation.Generally, however, a suitable distance between the focal point of theacoustic radiation and the fluid surface is in the range of about 1 toabout 15 times the wavelength of the speed of sound in the fluid, moretypically in the range of about 1 to about 10 times that wavelength,preferably in the range of about 1 to about 5 times that wavelength.

“Optional” or “optionally” means that the subsequently describedcircumstance may or may not occur, so that the description includesinstances where the circumstance occurs and instances where it does not.

The term “radiation” is used in its ordinary sense and refers toemission and propagation of energy in the form of a waveform disturbancetraveling through a medium such that energy is transferred from oneparticle of the medium to another without causing any permanentdisplacement of the medium itself. Thus, radiation may refer, forexample, to electromagnetic waveforms as well as acoustic vibrations.

The term “reservoir” as used herein refers to a receptacle or chamberfor containing a fluid. Thus, a fluid in a reservoir necessarily has afree surface, i.e., a surface that allows a droplet to be ejectedtherefrom. A reservoir may also be a locus on a substrate surface withinwhich a fluid is constrained or held. In some instances, a reservoir mayrepresent a portion, e.g., a fluid-transporting feature of amicrofluidic device.

The term “substantially” as in, for example, the phrase “substantiallyidentical volume,” refers to volumes that do not deviate by more than10%, preferably not more than 5%, more preferably not more than 1%, andmost preferably at most 0.1% from each other. Similarly, the phrase“substantially identical reservoirs” refers to reservoirs that do notdeviate in acoustic properties. For example, acoustic attenuations of“substantially identical reservoirs” deviate by not more than 10%,preferably not more than 5%, more preferably not more than 1%, and mostpreferably at most 0.1% from each other. Other uses of the term“substantially” involve an analogous definition.

The term “substrate” as used herein refers to any material having asurface onto which a sample—cellular or otherwise—may be placed andoptionally immobilized. The substrate may be constructed in any of anumber of forms, for example, wafers, slides, well plates, andmembranes. In addition, the substrate may be porous or nonporous as maybe required for deposition of a particular fluid. Suitable substratematerials include, but are not limited to, supports that are typicallyused for solid phase chemical synthesis, for example, polymericmaterials (e.g., polystyrene, polyvinyl acetate, polyvinyl chloride,polyvinyl pyrrolidone, polyacrylonitrile, polyacrylamide, polymethylmethacrylate, polytetrafluoroethylene, polyethylene, polypropylene,polyvinylidene fluoride, polycarbonate, and divinylbenzene styrene-basedpolymers), agarose (e.g., Sepharose®), dextran (e.g., Sephadex®),cellulosic polymers and other polysaccharides, silica and silica-basedmaterials, glass (particularly controlled pore glass (CPG) andfunctionalized glasses), ceramics, and such substrates treated withsurface coatings, such as microporous polymers (particularly cellulosicpolymers such as nitrocellulose), microporous metallic compounds(particularly microporous aluminum), antibody-binding proteins(available from Pierce Chemical Co., Rockford, Ill.), bisphenol Apolycarbonate, or the like. Additional information relating to the term“substrate” can be found in U.S. Patent Application Publication No.20020037579 to Ellson et al.

The term “surface modification” as used herein refers to the chemicaland/or physical: alteration of a surface by an additive or subtractiveprocess to change one or more chemical and/or physical properties of asurface or a selected site or region of a surface. For example, surfacemodification may involve (1) changing the wetting properties of asurface, (2) functionalizing a surface, i.e., providing, modifying, orsubstituting surface functional groups, (3) defunctionalizing a surface,i.e., removing surface functional groups, (4) otherwise altering thechemical composition of a surface, e.g., through etching, (5) increasingor decreasing surface roughness, (6) providing a coating on a surface,e.g., a coating that exhibits wetting properties that are different fromthe wetting properties of the surface, and/or (7) depositingparticulates on a surface.

The term “tissue” as used herein refers to an aggregation ofmorphologically similar cells and associated intercellular matter actingtogether to perform one or more specific functions in a multicellularorganism, e.g., a plant or an animal. When the tissue is from an animal,for example, the tissue may be muscle, nerve, epidermal, blood, orconnective.

The term “volatility” is used herein in its ordinary sense and refers tothe ability of a material to evaporate or become vaporized. For example,a “low volatility” fluid is less easily vaporized than a “highvolatility” fluid.

The invention generally provides a method for preparing a sample surfacefor analysis. The method involves first placing a sample surface indroplet-receiving relationship to a fluid-containing reservoircontaining an analysis-enhancing fluid. A droplet of theanalysis-enhancing fluid is ejected from the reservoir such that thedroplet is deposited on the sample surface at a designated site.Typically, but not necessarily, focused energy, e.g., acoustic energy,is employed to effect droplet ejection. Furthermore, the sample issubjected to conditions sufficient to allow the analysis-enhancing fluidto interact with the sample surface to render the sample surface at thedesignated site suitable for analysis. Optionally, sufficient energy isapplied to the designated site to ionize the sample surface and torelease sample molecules therefrom for analysis.

The invention may be used with any sample having a uniform or nonuniformsurface of any type. For instance, the sample may be comprised oforganic as well as inorganic materials. Examples of inorganic materialsinclude, but are not limited to metals, ceramics, and semiconductors.When the sample is comprised of an organic material, the material may beliving, nonliving, or dead.

Nevertheless, cellular samples are particularly suited for use with theinvention. Notably, a cellular sample may comprise nearly any type ofcell. For example, either or both eukaryotic cells and prokaryotic cellsmay be used. In some instances, cellular samples are obtained from amammal. Suitable cell types include, for example, blood cells, stemcells, endothelial cells, epithelial cells, bone cells, liver cells,smooth muscle cells, striated muscle cells, cardiac muscle cells,gastrointestinal cells, nerve cells, and cancer cells. Such cells may beprovided as a tissue sample. Alternatively, the cellular sample maycomprise cells originating from a cultured cell line or other cellculture.

The cellular sample may be employed in any of a number of forms. Thecellular sample is typically nonuniform in composition, and exhibits asubstantially planar surface for ease in array formation when aplurality of droplets is deposited on designated sites, as discussedbelow. In addition, cells are typically immobilized on a substratesurface. Thus, a substrate surface may be selected for facileimmobilization of cells. Such surfaces include, for example, acollagen-derivatized surface, dextran, polyacrylamide, nylon,polystyrene, and combinations thereof. In some instances, the surfacesare inherently cytophilic. In other instances, a cytophilic substratesurface is provided as a result of surface modification.

Cells may be immobilized on a substrate surface using conventionaltechniques known to those skilled in the art. For example, the cells maybe immobilized on a cytophilic substrate surface simply by contactingthe surface with the cells. In some instances, a centrifuge may be usedto produce a force sufficient to immobilize cells in a fluid on asubstrate surface. In other instances, cytometers known in the art maybe used. In still other instances, acoustic ejection, as described inU.S. Patent Application Publication Nos. 2002/0064808, 2002/0064809,2002/0094582, and 2002/0090720, may be carried out to place cells on asubstrate surface. In addition or in the alternative, the substratesurface may be coated with a layer of a cell-adhering substance, such ascollagen, alginate, agar, or other material, to immobilize the cells.When immobilization of cells in a contiguous layer is desired, thecell-adhering substance may be contiguously coated on the target region.When it is desirable to provide an immobilized array of cells, however,the cell-adhering substance may be present as an array of features on asubstrate surface. That is, an array of locations on a substrate surfacemay be coated with an appropriate material to form an array (e.g.,patterns such as lanes, checkerboards, spots, or others) so that cellsmay be spatially arranged at specific locations on the substratesurface.

When the cellular sample is a tissue sample, any of the aboveimmobilization methods may be used. In addition, immobilization oftissue samples may be accomplished by first freezing a relatively largesection of tissue. Thereafter, a slicing means such as a knife,microtome, or similar sectioning device is used to slice the frozentissue into sections. Next, a single section of the tissue is placedonto the substrate (e.g., a glass slide), and the section is allowed to“melt” on the substrate. Those skilled in the art will recognize otherimmobilization techniques that can be used as well.

In addition, the invention may involve the ejection of fluids ofvirtually any type and amount desired. The fluids may be aqueous and/ornonaqueous. Examples of fluids include, but are not limited to, aqueousfluids (including water per se and water-solvated ionic and non-ionicsolutions, organic solvents, and lipidic liquids); suspensions ofimmiscible fluids; and suspensions or slurries of solids in liquids.Thus, the fluid may contain, for example, biomolecules such as peptidesor nucleotides, enzymes, and/or cellular matter such as whole cells andcell extracts. Commonly, the fluid is selected to preferentiallyinteract with selected moieties on the compositionally nonuniform samplesurface. For example, if peptidic digestion is desired, then trypsin,pepsin, or other well-known compounds for peptidic digestion may beincluded in the fluid. When the invention is used for sample analysis,the fluid may be an analysis-enhancing fluid. That is, theanalysis-enhancing fluid may be employed to increase yield of usefulinformation during surface analysis. In some instances, theanalysis-enhancing fluid comprises an analysis-enhancing moiety and acarrier fluid. For example, the analysis-enhancing moiety may be a labelmoiety that is fluorescently, magnetically, or radioactively detectable.The carrier fluid in such cases may include, for example, combinationsof water, acetonitrile, alcohols such as ethanol, and ketones such asacetone.

The invention is particularly suited for instances in which a pluralityof droplets of one or more analysis-enhancing fluids is deposited on thesample surface. In some instances, the plurality of droplets isdeposited on the sample surface at the same designated site. Thistechnique provides control over the formation of the feature at thedesignated site. For example, if the droplets deposited at the samedesignated site contains different moieties, the concentration of thedifferent moieties that form the feature can be controlled. In addition,the deposition of droplets at a designated site may correct for anypotential deficiency in the presence of a required fluid at the site.

In addition or in the alternative, one or more droplets ofanalysis-enhancing fluid may be ejected onto the sample surface atdifferent designated sites. In some instances, the different designatedsites form an array. In such a case, because the analysis-enhancingfluid renders the designated sites more amenable for analysis, theanalysis of the array would result in the analytical “imaging” of thesample surface, as discussed below.

Focused acoustic ejection enables the preparation of arrays that mayhave a density substantially higher than that possible using other arraypreparation techniques, such as capillary microspotting andpiezoelectric techniques (e.g., using inkjet printing technology). Thatis, focused acoustic ejection allows for the preparation of arrays withnearly limitless densities of array elements (i.e., sites). In someinstances, the density is in the range of approximately 10 toapproximately 250,000 array elements (i.e., sites) per square centimeterof sample, typically in the range of approximately 400 to approximately100,000 array elements per square centimeter of sample surface. However,it must be emphasized that the present method enables preparation of farhigher density arrays as well, i.e., arrays comprised of at least about1,000,000 array elements per square centimeter of sample surface, oreven in the range of about 1,500,000 to 4,000,000 elements per squarecentimeter of sample surface. Also, the matrix can be precisely appliedto a sample spot in increments of one picoliter or less, allowing anunparalleled degree of precision

Designated sites may correspond to sample regions of specific analytes.Since different analysis-enhancing fluids may specifically enhance onetype of analyte over another, the experimenter could effectively analyzean impure sample. For example, one could deposit two analysis-enhancingfluids on two separate subregions of a single analyte region of asample, or on two or more regions of the same analyte. A region is anarea containing analyte that is not contiguous with an adjacent region.A subregion is an area of analyte within a non-contiguous region.

When an analysis-enhancing fluid is deposited on the sample surface, thesample is typically subjected to conditions effective to allow theanalysis-enhancing fluid to interact with the sample surface, renderingthe sample surface suitable for analysis. Depending on the type ofanalysis desired, any of a number of different types of interactions maytake place between the analysis-enhancing fluid and the sample surface.Thus, for example, when the analysis-enhancing fluid comprises ananalysis-enhancing moiety and a carrier fluid, the carrier fluid may beevaporated from the sample surface, thereby leaving theanalysis-enhancing moiety in a concentrated form on the sample surface.That is, evaporation of the carrier fluid may increase the localconcentration of the analysis-enhancing moiety to effect interactionbetween the analysis-enhancing moiety and the sample surface.

Depending on the type of analysis desired, any of a number of differenttypes of interaction may take place between the analysis-enhancingmoiety and the sample surface. For example, the analysis-enhancingmoiety may be selected to break down or digest the constituents of thesample surface. As another example, the analysis-enhancing moiety maybind with selective moieties on the sample surface, thereby renderingthe sample surface suitable for analysis. As a further example, theanalysis-enhancing fluid may be solidified on the sample surface.

Once appropriate interaction has taken place, the sample at thedesignated site may be analyzed. Any of a number of analyses may becarried out for the sample at the designated site or sites.Surface-specific properties may be measured by surface-specific physicaltechniques and physical techniques that are adapted to surfacecharacterization. Various physical surface characterization techniquesinclude, without limitation, diffractive techniques, spectroscopictechniques, microscopic surface imaging techniques, surface ionizationmass spectroscopic techniques, thermal desorption techniques, andellipsometry. It should be appreciated that these classifications arearbitrarily made for purposes of explication, and some overlap mayexist. Thus, for example, the inventive device may incorporate analyzingmeans such as microscopes, optical detectors, fluorescence detectors,magnetic detectors, radioactivity detectors, and combinations thereof.

The invention is particularly suited for mass spectrometry analysisapplications involving laser-assisted ionization and desorption. MALDIis an example of such applications. See Karas et al. (1988), “LaserDesorption Ionization of Proteins with Molecular Masses Exceeding 10,000Daltons,” Anal. Chem., 60:2299-2301. In addition, the invention may beemployed in conjunction with a variety of surface-based massspectrometric techniques other than MALDI. For example, one variant ofMALDI, called SELDI, uses affinity capture reagents such as antibodiesto collect samples from a complex mixture, which allows in situpurification of the analyte followed by conventional MALDI analysis. Insuch analytical methods, the invention may be used to apply sufficientenergy to the sites to ionize and release the sample molecules from thesample surface for analysis. The energy may be applied, for example, bybombarding the sites with photons (e.g., through use of an optionallaser), electrons, and/or ions. Ionization and release of samplemolecules may be enhanced through heating, directing focused acousticenergy to, and/or passing an electrical current through, at least onesite. Once released, the ions may be directed to a massanalyzer/detector. By collecting such mass spectrometric data for aplurality of sites, a compositional map of the sample surface can beconstructed. As discussed above, different regions of a single samplemay be co-crystallized or complexed with a variety of matrices tofacilitate the ionization of a particular component of interest.

For MALDI or SELDI-type analysis, the analysis-enhancing fluid comprisesa mass-spectrometry matrix material. Any of a number of photoabsorbingmatrix materials known in the art may be employed, and examples ofmatrix materials for sample analysis include, but are not limited to,6-aza-2-thiothymine, caffeic acid, 2,5-dihydroxybenzoic acid,α-cyano-4-hydroxy cinnamic acid, 3-hydroxypicolinic acid, and2-pyrazinecarboxylic acid, and combinations thereof. A plurality ofanalysis-enhancing fluids may be applied to an analyte to optimizeexperimental parameters, such as signal and reproducibility. Forexample, different sub-regions of a single sample could also be probedwith a variety of matrices to enhance a particular component ofinterest. In addition, certain mass-spectrometry matrix materials areparticularly suited for certain types of samples. As examples,3-hydroxypicolinic acid is commonly used for the analysis ofglycoproteins, 2-amino-5-nitropyridine is suited for nucleic aciddetection, and 6aza-2-thiothymine may be used to analyze proteins.

As discussed above, the accuracy and reliability of such massspectrometric techniques require control over the formation of thesample matrix. Thus, to provide control of analyte ionization anddesorption, it is preferred that features be formed in a consistentmanner. This typically requires the deposition of fluid droplets ofsubstantially identical size on a substrate. For example, if thesubstrate already contains a matrix material, sample droplets containingthe same concentration of analyte moieties and of a substantiallyidentical size may be deposited as an array on the substrate. As anotherexample, if the substrate already contains sample materials, identicallysized droplets of one or more matrix materials may be deposited onselected sites to form features that facilitate sample ionization anddesorption upon bombardment of laser photons. In either case, the matrixmaterial enables the absorption of laser energy to volatilize and ionizethe analyte, while preventing analyte decomposition by absorbingsignificant amounts of laser energy.

For mass spectrometric analysis using the invention, acoustic ejectionis employed to coat either an entire sample surface or only designatedsites thereon with a mass-spectrometry matrix material. Either or bothof the matrix materials and the analyte may thus be deposited on thesubstrate surface consistently and homogeneously from site to site. Ifeither the matrix material or the analyte is absent, or is present in aninappropriate quantity at a feature, proper ionization will not takeplace, thereby resulting in inoperative or suboptimal MALDI performance.For example, when fluids are deposited manually to form features on asubstrate or a sample surface, one can expect highly variable signalstrengths from the different individual features. In many cases, nosignal is detected. Moreover, manual deposition of fluid features doesnot typically enable the study of substructures in a sample wherein thesample features have a cross-sectional dimension of about 10-20 μm.

In contrast, the invention provides control over the formation of afeature at a designated site. For example, in the context of massspectrometry, the invention's ability to eject additional matrixmaterial to designated sites that lack sufficient matrix materialprovides fine control over the amount of matrix material present at adesignated site. In particular, acoustic ejection allows highlyreproducible quantities of MALDI matrix material, analyte, or anotherchemical entity to be deposited on regions of a substrate surface.Acoustic ejection additionally provides control over the formation ofthe sample/matrix complex on the sample surface. As described in U.S.Patent Application Publication No. 2002/0061258, employment of acousticejection to dispense fluids results in consistency of feature shape,droplet directionality, and ejected volume that is unmatched by printingmethods generally known in the art. Features containing matrix materialson the order of micrometers can be created. Due to the repeatability andprecision in placement of droplets through acoustic ejection, additionalmatrix material may be added to any desired feature site, e.g., tocorrect for any potential deficiency in the presence of a requiredfluid. That is, for any feature site, matrix material may beincrementally deposited to ensure that the amount of matrix material atthat feature site is optimized for data acquisition. In addition,because acoustic ejection allows for precise placement of ejecteddroplets, the location of matrix materials at the designated sites willbe known with a higher degree of confidence. As a result, there will beno need for the laser to probe a sample multiple times simply to locatethe complex. Increasing the frequency and success of experiments greatlyreduces the time for sample analysis, leading to greater samplethroughput.

When MALDI-type analysis is carried out at these sites, sufficientenergy is applied to the sites to ionize and release the samplemolecules from the sample surface for analysis. This may involvebombarding a designated site with photons through the use of an optionallaser. As discussed above, different regions of a single sample may beco-crystallized with a variety of matrices to facilitate the ionizationof a particular component of interest.

Furthermore, the invention is well suited for mass-spectrometricimaging. Such imaging has been described in detail in U.S. Pat. No.5,808,300 to Caprioli and in Stoeckli et al. (2001), “Imaging MassSpectrometry: A New Technology for the Analysis of Protein Expression inMammalian Tissues,” Nature Medicine 7(4):493-496. In essence, thesereferences describe the use of MALDI mass spectrometry to generateimages of samples according to one or more massto-charge ratios. Thatis, MALDI mass spectrometry is used to analyze the spatial arrangementof specific molecules within a tissue sample. The analysis involvesfirst preparing a test specimen by coating a thin sample layer with anenergy absorbent matrix. Then, the test specimen is probed with a laserbeam such that a first designated laser spot on the test specimenreleases sample molecules. The test specimen is then moved relative tothe laser beam and struck with the laser beam such that subsequentdesignated laser spots on the test specimen release additional samplemolecules. The atomic mass of the released sample molecules are measuredand mapped to determine the spatial arrangement of specific moleculeswithin the sample. It should be noted that for such mass spectrometricimaging applications, it is desirable to provide an analysis-enhancingfluid that contains mass-spectrometry matrix material that willco-crystallize with, or form crystals on, a sample surface whendeposited in small volumes. By careful application of small volumes,high-resolution maps of the composition of sample surfaces can begenerated.

A variety of techniques may be used to deposit small volumes ofanalysis-enhancing fluid on a sample surface. Typically, the inventionprovides a device or system that includes a reservoir adapted to containa fluid, an acoustic ejector, an acoustic ejector positioning means, anda sample positioning means. The acoustic ejector comprises an acousticradiation generator for generating acoustic radiation and a focusingmeans for focusing the acoustic radiation that is generated. The ejectorpositioning means is adapted for positioning the acoustic ejector inacoustic coupling relationship to the reservoir. The sample positioningmeans is adapted for positioning a sample such that a surface of thesample is in droplet-receiving relationship to the reservoir.

Although there is great flexibility in the construction of a suitablereservoir for the invention, some constructions are more suitable thanothers. As an initial matter, the material used in the construction ofthe reservoir should be compatible with the fluids contained therein.Thus, if it is intended that the reservoir contain an organic solventsuch as acetonitrile, polymers that dissolve or swell in acetonitrilewould be unsuitable for use in forming the reservoirs. For water-basedfluids, a number of materials are suitable for the construction of thereservoir; these include, but are not limited to, ceramics such assilicon oxide and aluminum oxide, metals such as stainless steel andplatinum, and polymers such as polyester and polytetrafluoroethylene.The reservoir is preferably detachable from the inventive device orsystem, but this is not a necessity. When more than one reservoir isneeded, a well plate may be used to contain fluids that are to beejected. In such a case, the reservoirs, or wells, of the well plate arepreferably substantially acoustically indistinguishable from oneanother. Also, unless it is intended that the ejector be submerged inthe fluid to be ejected, the reservoirs or well plates must haveacoustic transmission properties sufficient to allow acoustic radiationfrom the ejector to be conveyed to the surfaces of the fluids to beejected. Typically, this involves providing reservoir, or well, basesthat are sufficiently thin to allow acoustic radiation to traveltherethrough without unacceptable dissipation.

As alluded to above, a plurality of reservoirs may be provided, eachcontaining a different fluid. In such a case, focused acoustic energymay be applied in a manner effective to eject a droplet of fluid fromeach reservoir such that the droplets from the reservoirs are depositedon the sample surface. The droplets of different fluids may be depositedat a single designated site or at different designated sites.

Many commercially available well plates suitable are for use with theinvention, e.g., as a single reservoir unit that is comprised of aplurality of reservoirs, and may contain, for example, 96, 384, 1536,and 3456 wells per well plate. Manufacturers of suitable well plates foruse with the invention include Coming, Inc. (Corning, N.Y.) and GreinerAmerica, Inc. (Lake Mary, Fla.). The availability of such commerciallyavailable well plates, however, does not preclude the manufacture anduse of custom-made well plates that contain at least about 10,000 wells,or as many as 100,000 wells or more. In some instances, the reservoirsmay represent a portion of a microfluidic device, as discussed below.Microfluidic devices are available from ACLARA BioSciences, Inc.(Mountain View, Calif.), Caliper Technologies Corp. (Mountain View,Calif.), and Fluidigm Corp. (South San Francisco, Calif.). The combinedemployment of focused acoustic ejection and microfluidic devices isdiscussed in U.S. Patent Application Publication No. 20020125424 toEllson et al.

Because of the precision that is possible using the inventivetechnology, the above-described device or system may be used to ejectdroplets from a reservoir adapted to contain no more than about 100nanoliters of fluid, preferably no more than about 10 nanoliters offluid. In certain cases, the ejector may be adapted to eject a dropletfrom a reservoir adapted to contain about 1 to about 100 nanoliters offluid. This is particularly useful when it is desirable to ejectdroplets having small volumes, e.g., about 50 pL or less. In someinstances, the fluid to be ejected may contain rare or expensivebiomolecules. In such a case, the volume of the ejected droplet may beno greater than about 10 pL, preferably about 1 picoliter or less (e.g.,in the range of about 0.025 pL to about 1 pL), which corresponds todeposited droplet diameters of about 20 m or less.

FIG. 1 illustrates an embodiment of the inventive method for analyzing acellular sample through mass spectrometry. As with all figuresreferenced herein, in which like parts are referenced by like numerals,FIG. 1 is not to scale, and certain dimensions may be exaggerated forclarity of presentation. A device for acoustically depositing a fluid ona surface of a cellular sample is depicted in FIGS. 1 and 2. This deviceis similar in construction to the acoustic ejection device described inU.S. Patent Application Publication No. 2002037579. The device 11includes a plurality of reservoirs, i.e., at least two reservoirs,although only one reservoir may be required in some instances. A firstreservoir is indicated at 13, and a second reservoir is indicated at 15.Each is adapted to contain a fluid having a fluid surface, e.g., a firstfluid 14 and a second fluid 16 having fluid surfaces respectivelyindicated at 17 and 19. Typically, fluids 14 and 16 are different. Inorder to prepare the sample surface for mass spectrometric analysis,fluids 14 and 16 each comprise a mass-spectrometry matrix material.

The reservoirs are shown in their preferred construction and aresubstantially identical and acoustically indistinguishable. However,identical construction is not a requirement. In addition, the reservoirsare shown as separate removable components, but they may, if desired, befixed within a plate or other substrate. For example, the plurality ofreservoirs may comprise individual wells in a well plate, optimallyalthough not necessarily, arranged in an array. Each of the reservoirs13 and 15 is preferably axially symmetric as shown, having verticalwalls 21 and 23 extending upward from circular reservoir bases 25 and27, and terminating at openings 29 and 31, respectively, although otherreservoir shapes may be used. The material and thickness of eachreservoir base should be such that acoustic radiation may be transmittedtherethrough and into the fluid contained within the reservoirs.

The device also includes an acoustic ejector 33, comprised of anacoustic radiation generator 35 for generating acoustic radiation and afocusing means 37 for focusing the acoustic radiation at a focal pointwithin the fluid from which a droplet is to be ejected near the fluidsurface. As shown in FIGS. 1 and 2, the focusing means 37 may comprise asingle solid piece having a concave surface 39 for focusing acousticradiation, but the focusing means may be constructed in other ways asdiscussed below. The acoustic ejector 33 is thus adapted to generate andfocus acoustic radiation so as to eject a droplet of fluid from each ofthe fluid surfaces 17 and 19 when acoustically coupled to reservoirs 13and 15, and thus to fluids 14 and 16, respectively. The acousticradiation generator 35 and the focusing means 37 may function as asingle unit controlled by a single controller, or they may beindependently controlled, depending on the desired performance of thedevice. Typically, single ejector designs are preferred over multipleejector designs because accuracy of droplet placement and consistency indroplet size and velocity are more easily achieved with a singleejector. However, the invention is not limited to single ejectordesigns.

For the present invention, any of a variety of focusing means thatinclude curved surfaces or Fresnel lenses known in the art may beemployed in conjunction with the present invention. Such focusing meansare described in U.S. Pat. No. 4,308,547 to Lovelady et al. and U.S.Pat. No. 5,041,849 to Quate et al., as well as in U.S. PatentApplication Publication No. 2002037579. In addition, there are a numberof ways to acoustically couple the ejector to each individual reservoirand thus to the fluid therein. Although acoustic coupling can beachieved through direct contact with the fluid contained in thereservoirs, the preferred approach is to acoustically couple the ejectorto the reservoirs and reservoir fluids without allowing any portion ofthe ejector (e.g., the focusing means) to contact any of the fluids tobe ejected. To this end, the present invention provides an ejectorpositioning means for positioning the ejector in controlled andrepeatable acoustic coupling with each of the fluids in the reservoirsto eject droplets therefrom without submerging the ejector therein.

To avoid ejector submersion, direct or indirect contact is typicallyestablished between the ejector and the external surface of eachreservoir. When direct contact is used in order to acoustically couplethe ejector to each reservoir, it is preferred that the direct contactbe wholly conformal to ensure efficient acoustic energy transfer. Thatis, the ejector and the reservoir should have corresponding surfacesadapted for mating contact. Thus, if acoustic coupling is achievedbetween the ejector and reservoir through the focusing means, it isdesirable for the reservoir to have an outside surface that correspondsto the surface profile of the focusing means. Without conformal contact,efficiency and accuracy of acoustic energy transfer may be compromised.In addition, since many focusing means have a curved surface, the directcontact approach may necessitate the use of reservoirs that have aspecially formed inverse surface.

Optimally, acoustic coupling is achieved between the ejector and each ofthe reservoirs through indirect contact, as illustrated in FIGS. 1 and2. In the figures, an acoustic coupling medium 41 is placed between theejector 33 and the base 25 of reservoir 13, with the ejector andreservoir located at a predetermined distance from each other. Theacoustic coupling medium may be an acoustic coupling fluid, preferablyan acoustically homogeneous material in conformal contact with both theacoustic focusing means 37 and each reservoir. In addition, it isimportant to ensure that the fluid medium is substantially free ofmaterial having different acoustic properties than the fluid mediumitself. As shown, the first reservoir 13 is acoustically coupled to theacoustic focusing means 37 such that an acoustic wave generated by theacoustic radiation generator is directed by the focusing means 37 intothe acoustic coupling medium 41, which then transmits the acousticradiation into the reservoir 13.

In operation, reservoirs 13 and 15 of the device are each filled withfirst and second fluids 14 and 16, respectively, as shown in FIG. 1. Theacoustic ejector 33 is positionable by means of ejector positioningmeans 43, shown below reservoir 13, in order to achieve acousticcoupling between the ejector and the reservoir through acoustic couplingmedium 41. A cellular sample 45 in the form of a tissue section isplaced on a substrate 46, which is positioned above and in proximity tothe first reservoir 13 such that one surface of the tissue, shown inFIG. 1 as the underside surface 51, faces the reservoir and issubstantially parallel to the surface 17 of the fluid 14 therein. Oncethe ejector, the reservoir, and the tissue sample 45 are in properalignment, the acoustic radiation generator 35 is activated to produceacoustic radiation that is directed by the focusing means 37 to a focalpoint 47 near the fluid surface 17 of the first reservoir. As a result,droplet 49 is ejected from the fluid surface 17 onto a designated site55 on the underside surface 51 of the tissue sample. The ejected dropletmay be retained on the tissue surface through adsorption and/or othersurface effects. In some instances, the droplet or a nonvolatile portionthereof may be solidified on the tissue surface after contact; in suchan embodiment, it may be necessary to maintain the tissue at a lowtemperature, i.e., a temperature that results in droplet solidificationafter contact. When droplets are solidified on a tissue surface, it maybe desirable to raise all droplets to a temperature sufficient tointeract with the tissue surface so as to facilitate analysis.

Then, as shown in FIG. 2, a sample positioning means 50 repositions thecellular sample 45 on the substrate 46 over reservoir 15 in order toreceive a droplet therefrom at a second designated site 57. FIG. 2 alsoshows that the ejector 33 has been repositioned by the ejectorpositioning means 43 below reservoir 15 and in acoustically coupledrelationship thereto by virtue of acoustic coupling medium 41. Onceproperly aligned as shown in FIG. 2, the acoustic radiation generator 35of ejector 33 is activated to produce acoustic radiation that is thendirected by focusing means 37 to a focal point 48 within the fluid 16near the fluid surface 19, thereby ejecting droplet 53 onto the cellularsample at the second designated site 57. It should be evident that suchoperation is illustrative of how the inventive device may be used toeject a plurality of fluids from reservoirs in order to form a pattern,e.g., an array, on the cellular sample surface 51. It should besimilarly evident that the device may be adapted to eject a plurality ofdroplets from one or more reservoirs onto the same site of the cellularsample surface.

It will be appreciated that various components of the device may requireindividual control or synchronization to form an array on a samplesurface. For example, the ejector positioning means may be adapted toeject droplets from each reservoir in a predetermined sequenceassociated with an array to be prepared on a sample surface. Similarly,the sample positioning means for positioning the sample with respect tothe ejector may be adapted to position the sample surface to receivedroplets in a pattern or array thereon. Either or both positioningmeans, i.e., the ejector positioning means and the sample positioningmeans, may be constructed from, for example, motors, levers, pulleys,gears, a combination thereof, or other electromechanical or mechanicalmeans known to one of ordinary skill in the art. It is preferable toensure that there is a correspondence between sample movement, ejectormovement, and ejector activation to ensure proper array formation.

FIG. 3 schematically illustrates an ionization chamber of a massspectrometer. The ionization chamber 60 comprises a housing 62containing an ionization region 64, and an interface comprising acapillary 66 and an electrode 68 for attracting ions toward thecapillary 66. An energy applying means 70 in the form of a laser and asample positioning means 50 are provided as well. In operation, thesubstrate 46 is immobilized with respect to the positioning means 50. Asshown, the tissue sample 45 has been subjected to conditions effectiveto allow the analysis-enhancing fluid droplets 49 and 53 to interactwith sample surface 51 so as to render the sample surface suitable foranalysis at the designated sites 55 and 57, respectively. The electrode68 is charged, and once the first designated site 55 and the capillary66 are in proper alignment, the laser 70 is activated. As a result,ionized sample molecules, formed at the first designated site 55, arereleased into ionization region 64. The ions are then drawn toward theelectrode 68, enter the capillary 66, and are subsequently analyzed bythe mass analyzer/detector 72. Examples of such mass analyzers/detectorsinclude multipole detectors, e.g., quadruple detectors, which employ acharged surface that attracts or repels the ionized sample molecule. Theidentical procedure may be carried out for ionizing sample molecules atthe second designated site 57. Additional charged surfaces (not shown)may be placed in the ionization chamber to direct the trajectory ofions.

Thus, it should be evident that the acoustic ejection device illustratedin FIGS. 1 and 2 may share a common registration system with the massspectrometer illustrated in FIG. 3. This ensures that the samplepositioning means for the acoustic ejection device and the massspectrometer employ the identical sample movement system. In turn, laserirradiation will occur precisely at the designated sites. It should befurther evident that these procedures may be controlled usingcommercially available and/or customized software.

The system illustrated in FIGS. 1-3 provides a number of previouslyunrealized advantages in the art of mass spectrometry imaging. Forexample, the invention may be used in the analysis and imaging ofpeptides and proteins in tumors and other abnormal tissue. With theaccuracy and precision associated with acoustic droplet deposition, theanalysis may quantify or compare the levels of specific proteins thatare more highly expressed in tumors, and/or those that are diminished inexpression, relative to normal tissue. Without using acoustic depositionof mass spectrometry matrix materials, accurate location andquantitation of the proteins may not be possible. Furthermore, theinvention greatly improves mass spectrometric imaging that may provide agreater understanding of other types of protein expression, e.g., thatassociated with Parkinson's disease, as well as of drugs and metabolitesin tissue.

Furthermore, acoustic deposition increases the spatial resolution ofMALDI imaging because the diameter of the fluid droplets deposited canbe controlled to approach the diameter of the laser beam used to scanthe sample. This diameter is typically about 2 to about 25 μm. This isimportant because analysis-enhancing fluids may liberate samplemolecules from the sample surface to be analyzed. If droplets exceedingthe diameter of the laser focal spot are used, liberated componentmolecules from different regions of a tissue sample may “mix” and resultin cross-contamination. This mixing creates inaccurate or false massspectrometric images that do not reflect the actual composition of thesample surface. In short, use of excessively large droplets may causecomponent molecules to migrate and decrease the spatial resolution ofthe imaging. Since the component molecules within each droplet may befreely transported anywhere within each droplet, droplet size mayeffectively dictate image resolution.

In addition, the invention also provides a method for selectivelydepositing an analysis-enhancing fluid on a nonuniform sample surfacethat exhibits variations in a surface characteristic, wherein thesurface characteristic corresponds to desirability for receiving ananalysis-enhancing fluid. The surface characteristic corresponding todesirability for receiving analysis-enhancing fluid is typically acompositional or a morphological characteristic. Once a site on thesample surface has been selected according to the surface characteristicat the site, focused radiation, typically acoustic radiation, is appliedin a manner effective to eject a droplet of the analysis-enhancing fluidfrom a reservoir. In some instances, the sample may exhibit variationsin a plurality of surface characteristics, wherein each characteristiccorresponds to desirability for receiving a different analysis-enhancingfluid. In such a case, droplets of different analysis-enhancing fluidsmay be deposited on the sample surface at the corresponding selectedsites.

Optionally, the sample at the selected site may be analyzed. Typicalanalytical techniques that benefit from the invention include, but arenot limited to, mass spectrometry, structural analysis, and microscopy.In addition, the invention is also particularly suited for use withsurface imaging techniques. In some instances, a sample surface isimaged so as to map the variations in a surface characteristic, and oneor more sites are selected on the sample surface using the results ofthe surface imaging. Preferably, surface imaging results in theproduction of a digital image. Further optionally, the image contrast ofthe variations in the surface characteristic is increased before thesurface is imaged. This may involve, for example, staining the samplesurface such that regions exhibiting the desired surface characteristicare more readily resolved by an imaging means.

Typically, though not necessarily, the inventive method may be carriedout using a device or system similar to that described above adapted forselectively depositing an analysis-enhancing fluid on a surface of asample. As discussed above, provided are a reservoir containing ananalysis-enhancing fluid, an acoustic ejector comprising an acousticradiation generator for generating acoustic radiation and a focusingmeans for focusing the acoustic radiation generated, and a means forpositioning the acoustic ejector in acoustic coupling relationship tothe reservoir. Also provided is a means for selecting at least one siteon the sample surface for deposition of analysis-enhancing fluidthereon. Site selection is carried out according to a surfacecharacteristic that corresponds to desirability for receiving theanalysis-enhancing fluid. The system also includes a means forpositioning the sample such that the selected site or sites arepositioned in droplet-receiving relationship to the reservoir. Thesample positioning means may be adapted to controllably position thesample such that when a plurality of sites on the sample surface isselected, the selected sites are successively placed indroplet-receiving relationship to the reservoir. Optionally, a means foranalyzing the composition of the sample at the selected site may also beprovided.

FIGS. 4-8 illustrate an embodiment of the inventive method forselectively depositing and analyzing a cellular sample through massspectrometry. FIG. 4 illustrates an exemplary cellular sample 45 placedon a substrate 46 for use with the invention. As depicted, the sample 45is a tissue sample having a substantially planar and nonuniform surface51. As shown, the surface 51 includes two distinct regions, the firstregion 51A depicted without shading, and the second region 51B, depictedwith shading. Region 51A exhibits a first surface characteristic thatcorresponds to desirability for receiving a first analysis-enhancingfluid 14 (shown in FIGS. 3 and 4), and region 51B exhibits a secondsurface characteristic that corresponds to desirability for receiving asecond analysis-enhancing fluid 16 (shown in FIGS. 3 and 4).

In order to select sites on the sample according to desirability forreceiving analysis-enhancing fluid, it may be necessary to image thesample surface. As shown in FIG. 5, a combination unit 80 is providedthat serves as a selecting means and a controller. The combination unitincludes an imaging means 82 adapted to produce a digital image of thesample surface 51. The digital image of the sample surface captured bythe imaging means 82 is stored and optionally analyzed in thecombination unit 80. As a result, one or more sites on the samplesurface 51 may be selected according to the surface characteristics atthe site or sites. This selection data, in turn, may be used in a systemto selectively deposit fluids onto the sample surface at the sites. Asdepicted, once an image of the sample surface 51 is obtained, thecombination unit, acting as a selecting means, will determine that site55 in region 51A is desirable for receiving the first analysis-enhancingfluid 14 and site 57 in region 51B is desirable for receiving the secondanalysis-enhancing fluid 16.

An exemplary device for acoustically depositing a fluid on a surface ofa cellular sample is shown in FIGS. 6 and 7. The device is similar inconstruction to the acoustic ejection device depicted in FIGS. 1 and 2described in U.S. Patent Application Publication No. 20020037579 toEllson et al. As surface characteristics of regions 51A and 51B aredifferent, fluids 14 and 16, selected for deposition on regions 51A and51B, respectively, are different as well. In order to prepare the samplesurface for mass spectrometric analysis, fluids 14 and 16 each comprisea mass spectrometry matrix material.

In operation, reservoirs 13 and 15 of the device are each filled withfirst and second analysis-enhancing fluids 14 and 16, respectively, asshown in FIG. 6. The combination unit 80 is provided to achievecorrespondence between sample movement, ejector movement, and ejectoractivation. The ejector positioning means 43 positions the acousticejector 33 below reservoir 13 and to achieve acoustic coupling betweenthe ejector and the reservoir through acoustic coupling medium 41.Cellular sample 45 is positioned by sample positioning means 50 aboveand in proximity to the first reservoir 13 such that the exposed surface51 of the tissue faces the reservoir and is substantially parallel tothe surface 17 of the fluid 14 therein. Site 55 in region 51A is placedin position to receive a droplet of the first analysis-enhancing fluid14 from reservoir 13. Once the ejector, the reservoir, and the tissuesample 45 are in proper alignment, the combination unit 80 activatesacoustic radiation generator 35 to produce acoustic radiation. Thefocusing means 37 directs the generated acoustic radiation to a focalpoint 47 near the fluid surface 17 of the first reservoir. As a result,droplet 49 is ejected from the fluid surface 17 onto the first selectedsite 55 on surface 51 of the tissue sample.

Then, as shown in FIG. 7, a sample positioning means 50 repositions thecellular sample 45 on the substrate 46 over reservoir 15 in order toreceive a droplet therefrom at the second selected site 57. In addition,the ejector 33 has been repositioned by the ejector positioning means 43below reservoir 15 and in acoustically coupled relationship thereto byvirtue of acoustic coupling medium 41. With proper alignment droplet 53is ejected onto the cellular sample at the second selected site 57. FIG.8 schematically illustrates the ionization chamber of FIG. 3 as adaptedfor use with the sample prepared using the device depicted in FIGS. 6and 7. The mass ionization chamber may also employ a common registrationsystem with the acoustic ejection device illustrated in FIGS. 6 and 7and the mass spectrometer illustrated in FIG. 5.

The surface characteristic corresponding to desirability for receivinganalysis-enhancing fluid is typically a compositional or morphologicalcharacteristic. Such characteristics may be optically and/oracoustically detectable. The detectability may optionally be enhanced bystaining or through the use of acoustic contrast enhancement fluids.

Thus, in some embodiments, the inventive system may include a selectingmeans comprised of a means for imaging the sample surface so as to mapthe variations in the surface characteristic, and a means for selectingat least one site on the sample surface using the results produced bythe imaging means. The imaging means is typically adapted to produce adigital image and may, for example, employ a scanner or a camera. Inother instances, an image of the sample surface that maps the variationsin the surface characteristic is produced without an imaging means. Ineither case, the selecting means may select sites for analysis-enhancingfluid deposition when the surface characteristic is above a thresholdlevel and/or when the surface characteristic is within a predeterminedrange. The selecting means may employ imaging technologies known in theart. A variety of imaging software, firmware, and hardware arecommercially available, as well as microscopes and other imagemagnification devices suitable for providing more detailed resolution ofsurface inhomogeneities. Optionally, the image contrast of thevariations in the surface characteristic is increased before the surfaceis imaged. This may involve, for example, staining or labeling thesample surface such that regions exhibiting the desired surfacecharacteristic are more readily resolved by an imaging means.

In some instances, the sample may exhibit variations in a plurality ofsurface characteristics, wherein each characteristic corresponds todesirability for receiving a different analysis-enhancing fluid. In sucha case, droplets of different analysis-enhancing fluids may be depositedon the sample surface at the corresponding selected sites. In addition,different analysis-enhancing fluids may be deposited on the samplesurface at the same site. In some instance, droplets of a firstanalysis-enhancing fluid may be sparsely deposited on an array ofsurface sites so as to survey the surface and to provide a “big picture”overview of the surface characteristics of interest. From the “bigpicture” overview, one may determine whether and optionally how todeposit additional droplets of the same and/or differentanalysis-enhancing fluid to form a high-density array of on the samplesurface.

The analysis-enhancing fluid may be selected according to the type ofanalysis desired, preferably to increase the yield of useful informationduring surface analysis. In some instances, the analysis-enhancing fluidcomprises a label moiety, such as one or more selected from the groupconsisting of a fluorescent moiety, a magnetic moiety, and a radioactivemoiety. However, it should be noted that label moieties do not have tobe employed in conjunction with the analysis-enhancing fluid; such labelmoieties may be used separately from the use of analysis-enhancingfluid. In addition or in the alternative, the analysis-enhancing fluidmay contain biomolecules (such as peptides or nucleotides) and/orcellular matter (such as whole cells and cell extracts). Often, theanalysis-enhancing fluid is selected to preferentially interact withselected moieties on a compositionally nonuniform sample surface. Forexample, if peptidic digestion is desired, then trypsin, pepsin, orother well-known compounds for peptidic digestion may be included in thefluid. Thus, an enzymatic biomolecule such as a protease is provided toenhance breakdown of the corresponding substrate moiety on or near thesample surface.

In addition, the analysis-enhancing fluid may comprise ananalysis-enhancing moiety and a carrier fluid. The carrier fluid in suchcases may include, for example, combinations of water, acetonitrile,alcohols (such as ethanol), and ketones (such as acetone). Thus, itshould be apparent that the invention may involve the ejection of fluidsof virtually any type and amount desired, including fluids that do notenhance analysis, although at least one analysis-enhancing fluid istypically used. The fluids may be aqueous and/or nonaqueous. Examples offluids include, but are not limited to, aqueous fluids (including waterper se and water-solvated ionic and nonionic solutions, organicsolvents, and lipidic liquids), suspensions of immiscible fluids, andsuspensions or slurries of solids in liquids. Evaporation of the carrierfluid increases the local concentration of the analysis-enhancing moietyto effect interaction between the analysis-enhancing moiety and thesubstrate surface. Depending on the type of analysis desired, any of anumber of different types of interaction might take place between theanalysis-enhancing moiety and the sample surface. For example, theanalysis-enhancing moiety may be selected to break down or digest theconstituents of the sample surface. As another example, theanalysis-enhancing moiety may bind with selective moieties on the samplesurface, thereby rendering the substrate surface suitable for analysis.Thus, analysis-enhancing fluids may enhance the suitability of a sampleto be analyzed by modifying the sample surface.

Means other than mass spectrometry for analyzing the sample may beprovided as well. Such analyzing means may include, for example, anoptical detector, a radiation detector, and/or a magnetic detector. Insome instances, the invention may be used to enhance analysis ofinorganic material. One example in which this may be carried out is toselectively deposit a particular etchant on an alloy surface to etch aparticular phase of the alloy. As another example, the invention mayenhance strain analysis. For instance, U.S. Pat. No. 6,327,030 to Ifjuet al. describes a method for measuring strain on a substrate material.The method involves preparing the substrate material for strainmeasurement by applying a luminescent coating to a substrate material,curing the coating, and illuminating the coating with excitationillumination. Then, characteristics of luminescent light emanating fromthe coating are measured in order to determine strain on the substratematerial. The characteristics of luminescent light emanating from thecoating are related to strain on the substrate material due, at least inpart, to a relationship between the amount of strain on the substratematerial and the morphology of cracks in the coating. The presentinvention provides a convenient technique to improve such a strainmeasurement method. Since it is known that some materials exhibitpreferential strain proliferation when they possess certainmorphological characteristics, e.g., surface scratches and texturing,the coating may be selectively applied only to regions on the substratethat exhibit such morphological characteristics for analysis. In thisway, excess coating is not wasted through its application to regionswhere such characteristics are absent.

It should be noted that the invention may be advantageously used incombination with surface mapping technologies, such as those describedin U.S. Pat. No. 5,808,300 to Caprioli and in U.S. Patent ApplicationPublication No. 2002/0171037 to Ellson et al. For example, FIG. 9schematically depicts tissue sample 45 on a substrate 46. The exposedsample surface 51 is nonuniform. As shown, the surface 51 is comprisedof a first region 51A circumscribing a second region 51B. The firstregion 51A may be, for example, comprised of lean tissue and the secondregion 51B comprised of lipid tissue. In such a situation, it may bedesirable to place an analysis-enhancing fluid on only one of theregions. That is, the sites for fluid deposition may be selectedaccording to lipid and/or peptide content at the site. As lipid tissueand lean tissue are typically optically distinguishable from oneanother, fluid deposition in this instance would involve selecting siteson the surface according to the optical characteristics displayed (i.e.,at the sites associated with either lipid or lean tissue).

The invention provides a method that combines the mapping technologiesas discussed above with the advantages of enhancement of analysis at aselected region. An example of such combined use is depicted in FIG. 9,wherein droplets of a first analysis-enhancing fluid, indicated at 49,are deposited at a first array of sites on the sample surface 51, anddroplets of a second analysis-enhancing fluid, indicated at 53, aredeposited at a second array of sites on the sample surface 51. The firstfluid enhances peptide analysis while the second fluid enhances lipidanalysis. The first fluid is deposited in both regions 51A and 51B,while the second fluid is deposited only within region 51B. Thus, byanalyzing the sample at each site having the first fluid depositedthereon, a map of peptide composition of the overall sample surface canbe constructed. Similarly, by analyzing the sample at each site havingthe second fluid deposited thereon, the lipidic region 51B may beselectively analyzed.

As discussed above, the invention may involve the use ofanalyte-enhancing fluids, such as those containing a mass-spectrometrymatrix material dissolved in a solvent. When such a solution is ejectedfrom a reservoir toward a sample surface, the solution should contactthe surface and be subjected to conditions sufficient for the matrixmaterial to interact with and/or co-crystallize with the sample surfaceto render the sample surface suitable for analysis.

One factor that determines the quality of matrix crystals is the rate ofevaporation of the solvent from a mass-spectrometry matrix solution.Given various volumes of fluid solutions having the same shape, thesolvent evaporation rates for smaller fluid volumes are faster than forlarger fluid volumes, because the surface-to-volume ratio increases asfluid volume decreases. For example, a spherical droplet having a 20 μmdiameter will have five times the surface-to-volume ratio of a droplethaving a 100 μm diameter. Accordingly, the smaller droplet would lose agreater fraction of its total volume per unit time than the largerdroplet.

Typically, one of ordinary skill in the art would select a highlyvolatile solvent for use with a mass-spectrometry matrix to form amass-spectrometry analysis-enhancing fluid so as to facilitate rapidevaporation of the solvent and co-crystallization of the matrix materialwith the sample surface. Similarly, low volatility solvents such asdimethylsulfoxide (DMSO) and glycerol are avoided in MALDI-TOFapplications. It has recently been discovered that when small-volumedroplets containing a mass-spectrometry matrix material dissolved in ahighly volatile solvent are ejected from a reservoir, the matrixmaterial may not co-crystallize properly with the sample surface. Inextreme cases, the matrix material will not co-crystallize at all withthe sample surface. For example, a typical MALDI matrix solution for thestudy of protein samples contains a 1:1 mixture of water andacetonitrile containing 0.2% trifluoroacetic acid. The solution containssinapinic acid at a near saturation concentration of 40 mg/mL. When sucha solution was spotted onto a typical gold substrate containing a tissuesample at volumes of 30 picoliters or less, the signal was not adequatefor protein identification. Therefore, it is likely that an inadequateamount of sample was co-crystallized with the matrix to allow signalacquisition, and that the amount of deposited matrix was inadequate. Itis further likely that these co-crystallization problems were caused byexcessive evaporation of the solvent while the droplets were travelingfrom the reservoir to the sample surface. Even when droplets containinga highly volatile solvent contact the surface, such droplets may nothave sufficient time to penetrate and interact with the surface beforethe solvent evaporates. Thus, the invention also provides for methodsand techniques to slow evaporation rates for small droplets ofmass-spectrometric matrix solutions so as to allow adequate analyteco-solvation and crystallization.

In this regard, the invention also provides for a method for preparing asample surface for analysis that involves the use of ananalysis-enhancing fluid comprising an analysis-enhancing moiety and acarrier fluid, wherein the carrier fluid is comprised of a lowvolatility solvent that has a boiling point greater than 100° C. at apressure of 1 atmosphere. A sample is placed such that a surface thereofis in droplet-receiving relationship to a reservoir containing theanalyte-enhancing fluid. Once a droplet of the analysis-enhancing fluidis ejected from the reservoir and deposited on the sample surface at adesignated site, the sample is subjected to conditions sufficient toallow the analysis-enhancing fluid to interact with the sample surfaceto render the sample surface suitable for analysis. A low volatilitysolvent is employed to ensure that the analysis-enhancing moiety, e.g.,a mass-spectrometry matrix material, remains sufficiently solvated bythe solvent so as to retain its ability to interact with the samplesurface, so as to be suitable for analysis. As solvent volatility isgenerally correlated to its ability to slow the overall evaporation rateof a droplet of fluid, solvents lower in volatility may be selected fordroplets lower in volume. For example, solvents having boiling points ofat least 120° C., 150° C., and 180° C. at a pressure of 1 atmosphere maybe selected for droplets of decreasing lower volumes. Exemplary solventshaving low volatility include, but are not limited to, dimethylsulfoxide(DMSO), glycerol, propylene carbonate, valeronitrile, malononitrile,sulfolane, 3-methylsulfolane, and higher alcohols and ketones.

Optionally, the carrier fluid may comprise a mixture of high and lowvolatility solvents. The proportion of the solvents may be selectedaccording the desired performance. For mass spectrometry analysisdescribed herein using acoustic ejection, the carrier fluid typicallycontains the high volatility solvent at a higher concentration than thelow volatility solvent. Preferably, the carrier fluid contains at leastabout 75% of the low volatility solvent. More preferably, the carrierfluid contains at least about 90% of the low volatility solvent.Optimally, the carrier fluid contains at least about 98% of the lowvolatility solvent.

In addition, other techniques may be used to control the evaporation ofthe solvent. For example, in order to accelerate the evaporation of alow volatility solvent on a sample surface, one may subject the sampleto a higher temperature and/or a lower pressure. For samples kept underordinary laboratory conditions, this may involve subjecting the sampleto a temperature greater than 25° C. or a pressure lower than about 1atmosphere. Similarly, one may subject a sample to a lower temperatureand/or higher pressure to hinder evaporation of a high volatilitysolvent on a sample surface. For samples kept under ordinary laboratoryconditions, this may involve subjecting the sample to a temperature ofless than 25° C. and/or a pressure greater than 1 atmosphere. Thus, itshould be apparent to those of ordinary skill in the art that controlover solvent evaporation relates to the partial pressure of the solvent.In order to ensure that a solvent does not evaporate too quickly, asample having an analysis-enhancing fluid comprised of a massspectrometry matrix material and the solvent may be subjected to anatmosphere that is at least about 30% saturated with the carrier fluidto allow the analysis-enhancing fluid to interact with the samplesurface and to render the sample surface suitable for analysis. To slowevaporation of the solvent further, the atmosphere may be at least about50%, 75%, or 90% saturated with the carrier fluid.

Those of ordinary skill in the art will also recognize that thevolatility of a fluid is a relative measure that depends on both thecomposition of the fluid and the environment to which the fluid isexposed. In general, water at 25° C. in an overall environment pressureof 1 atmosphere (760 mm/Hg) may sometimes be considered a fluid ofneither high nor low volatility. The saturated vapor pressure for waterin such an environment is about 23.76 mm/Hg. If one defines that thevolatility quotient of the a fluid as the ratio of the environmentalpressure to the saturated vapor pressure the fluid, the volatilityquotient for water in such an environment is about 30. Using this wateras a reference fluid, then, a high volatility fluid would havevolatility quotient less than about 30 and a low volatility fluid wouldhave a volatility quotient greater than about 30. For the presentinvention, a variety of volatility quotients are suitable, e.g., 30, 50,75, 100.

Variations of the present invention will be apparent to those ofordinary skill in the art. For instance, one of ordinary skill in theart would recognize that the volatility of a fluid mixture may changeover time as more volatile components escape from the mixture. Theskilled artisan will also recognize that Rayleigh's Law may apply tosuch mixtures. For example, a droplet comprised of 50% water, 48%acetonitrile, and 2% DMSO will have a higher initial vapor pressure thana droplet comprised of pure water. As acetonitrile is volatilized fromsuch a droplet, the DMSO concentration will rise, and the droplet mayeventually have a lower vapor pressure than a droplet comprised of purewater. Ultimately, the equilibrium fluid concentration of the dropletwill be 30% water and 70% DMSO, depending on the relative humidity ofthe atmosphere in contact with the sample surface

It is to be understood that, while the invention has been described inconjunction with the preferred specific embodiments thereof, theforegoing description is intended to illustrate and not limit the scopeof the invention. Other aspects, advantages, and modifications withinthe scope of the invention will be apparent to those skilled in the artto which the invention pertains.

All patents, patent applications, and publications mentioned herein arehereby incorporated by reference in their entireties.

1. A method for preparing a sample surface for analysis, comprising: (a)providing a reservoir containing an analysis-enhancing fluid, whereinthe analysis-enhancing fluid is comprised of an analysis-enhancingmoiety and a carrier fluid, and the carrier fluid is comprised of a lowvolatility solvent that has a boiling point greater than 100° C. at apressure of 1 atmosphere; (b) placing a surface of a sample indroplet-receiving relationship to the fluid-containing reservoir; (c)applying focused acoustic energy in a manner effective to eject adroplet of the analysis-enhancing fluid from the reservoir such that thedroplet is deposited on the sample surface at a designated site; and (d)subjecting the sample to conditions sufficient to allow theanalysis-enhancing fluid to interact with the sample surface to renderthe sample surface suitable for analysis.
 2. The method of claim 1,wherein the carrier fluid further comprises a high volatility solventthat has a boiling point less than 100° C. at a pressure of 1atmosphere.
 3. The method of claim 2, wherein the carrier fluid containsthe high volatility solvent at a higher concentration than the lowvolatility solvent.
 4. The method of claim 1, wherein theanalysis-enhancing moiety comprises a mass spectrometry matrix material.5. The method of claim 4, wherein the mass spectrometry matrix materialis a photoabsorbing matrix material.
 6. The method of claim 1, whereinthe analysis-enhancing fluid comprises a label moiety.
 7. The method ofclaim 1, wherein the analysis-enhancing fluid comprises a biomolecule.8. The method of claim 1, wherein the low volatility solvent has aboiling point greater than 120° C. at a pressure of 1 atmosphere.
 9. Themethod of claim 8, wherein the low volatility solvent has a boilingpoint greater than 150° C. at a pressure of 1 atmosphere.
 10. The methodof claim 9, wherein the low volatility solvent has a boiling pointgreater than about 180° C. at a pressure of 1 atmosphere.
 11. The methodof claim 1, wherein the sample is a cellular sample.
 12. The method ofclaim 11, wherein the cellular sample is selected from a tissue sample,a cell culture, a single cell, or a plurality of cells immobilized on asubstrate surface.
 13. The method of claim 1, wherein the sample surfaceis substantially planar.
 14. The method of claim 1, wherein step (c) isrepeated such that a plurality of droplets is deposited on the samplesurface.
 15. The method of claim 14, wherein the plurality of dropletsis deposited on the sample surface at the same designated site.
 16. Themethod of claim 14, wherein the plurality of droplets is deposited onthe sample surface at different designated sites.
 17. The method ofclaim 16, wherein the different designated sites form an array.
 18. Themethod of claim 1, wherein the sample surface exhibits variations in asurface characteristic that correspond to desirability for receiving ananalysis-enhancing fluid, and the designated site is selected accordingto the surface characteristic at the site.
 19. The method of claim 18,wherein the surface characteristic is a compositional and/ormorphological characteristic.
 20. The method of claim 18, wherein thesurface characteristic is optically and/or acoustically detectable. 21.The method of claim 1, wherein step (a) comprises providing a pluralityof reservoirs, each containing a different analysis-enhancing fluid, andstep (c) comprises applying focused acoustic energy in a mannereffective to eject a droplet of fluid from each reservoir such that thedroplets from the reservoirs are deposited on at least one designatedsite on the sample surface.
 22. The method of claim 21, wherein dropletsof different analysis-enhancing fluids are deposited on the samplesurface at the same designated site.
 23. The method of claim 21, whereinthe droplets are deposited on the sample surface at different designatedsites.
 24. The method of claim 23, wherein the different designatedsites form an array.
 25. The method of claim 23, wherein the differentdesignated sites are selected according to variations in a surfacecharacteristic of the sample.
 26. The method of claim 1, furthercomprising, after step (d), (e) analyzing the sample at the selectedsite.
 27. The method of claim 26, further comprising, after step (d) andbefore step (e), (d′) applying effective energy to the designated siteto release sample molecules from the sample surface for analysis. 28.The method of claim 27, wherein step (d′) comprises bombarding thedesignated site with photons, electrons, ions, or combinations thereof.29. The method of claim 27, wherein step (d′) comprises heating,directing focused acoustic energy to, and/or passing an electricalcurrent through the designated site.
 30. The method of claim 26, whereinstep (e) comprises performing mass spectrometric analysis on the samplemolecules.
 31. A method for preparing a sample surface for analysis,comprising: (a) providing a reservoir containing an analysis-enhancingfluid, wherein the analysis-enhancing fluid is comprised of ananalysis-enhancing moiety and a carrier fluid, and the carrier fluid iscomprised of a low volatility solvent that has a boiling point greaterthan 100° C. at a pressure of 1 atmosphere; (b) placing a surface of asample in droplet-receiving relationship to the fluid-containingreservoir; (c) ejecting a droplet of the analysis-enhancing fluid fromthe reservoir such that the droplet is deposited on the sample surfaceat a designated site, wherein the ejected droplet has a volume of nogreater than about 50 pL; and (d) subjecting the sample to conditionssufficient to allow the analysis-enhancing fluid to interact with thesample surface to render the sample surface suitable for analysis. 32.The method of claim 31, wherein the volume of the ejected droplet is nogreater than about 10 pL.
 33. The method of claim 32, wherein the volumeof the ejected droplet is no greater than about 1 pL.
 34. The method ofclaim 33, wherein the volume of the ejected droplet is in the range ofabout 0.025 pL to about 1 pL.
 35. The method of claim 31, wherein step(c) is carried out by applying focused acoustic energy to theanalysis-enhancing fluid contained in the reservoir in a mannereffective to eject a droplet therefrom.
 36. The method of claim 35,further comprising, after step (d), (e) analyzing the sample at theselected site.
 37. The method of claim 36, further comprising, afterstep (d) and before step (e), (d′) applying effective energy to thedesignated site to release sample molecules from the sample surface foranalysis.
 38. The method of claim 37, wherein step (d′) comprisesbombarding the designated site with photons, electrons, ions, orcombinations thereof.
 39. The method of claim 36, wherein step (e)comprises performing mass spectrometric analysis on the samplemolecules.
 40. The method of claim 36, wherein the sample is a cellularsample.
 41. A method for preparing a sample surface for massspectrometry analysis, comprising: (a) providing a reservoir containingan analysis-enhancing fluid, wherein the analysis-enhancing fluid iscomprised of a mass spectrometry matrix material and a carrier fluid,and the carrier fluid is comprised of a low volatility solvent that hasa boiling point greater than 100° C. at a pressure of 1 atmosphere; (b)placing a surface of a sample in droplet-receiving relationship to thefluid-containing reservoir; (c) ejecting a droplet of theanalysis-enhancing fluid from the reservoir such that the droplet isdeposited on the sample surface at a designated site; and (d) subjectingthe sample to conditions sufficient to allow the analysis-enhancingfluid to interact with the sample surface to render the sample surfacesuitable for analysis.
 42. The method of claim 41, wherein the massspectrometry matrix material is a photoabsorbing matrix material. 43.The method of claim 42, wherein step (c) is carried out by applyingfocused acoustic energy to the analysis-enhancing fluid contained in thereservoir in a manner effective to eject a droplet therefrom.
 44. Themethod of claim 43, further comprising, after step (d), (d′) bombardingthe designated site with photons to release sample molecules from thesample surface for analysis.
 45. The method of claim 44, furthercomprising, after step (d′), (e) performing mass spectrometric analysison the sample molecules.
 46. The method of claim 42, wherein the sampleis a cellular sample.
 47. The method of claim 41, wherein step (d)comprises subjecting the sample to a temperature greater than 25° C. 48.The method of claim 41, wherein step (d) comprises subjecting the sampleto a pressure lower than about 1 atmosphere.
 49. A method for preparinga sample surface for mass spectrometry analysis, comprising: (a)providing a reservoir containing an analysis-enhancing fluid, whereinthe analysis-enhancing fluid is comprised of a mass spectrometry matrixmaterial and a carrier fluid, and the carrier fluid is comprised of ahigh volatility solvent that has a boiling point lower than 100° C. at apressure of 1 atmosphere; (b) placing a surface of a sample indroplet-receiving relationship to the fluid-containing reservoir; (c)ejecting a droplet of the analysis-enhancing fluid from the reservoirsuch that the droplet is deposited on the sample surface at a designatedsite; and (d) subjecting the sample to a temperature lower than 25° C.and/or a pressure greater than 1 atmosphere to allow theanalysis-enhancing fluid to interact with the sample surface and torender the sample surface suitable for analysis.
 50. The method of claim49, wherein the mass spectrometry matrix material is a photoabsorbingmatrix material.
 51. The method of claim 49, wherein step (c) is carriedout by applying focused acoustic energy to the analysis-enhancing fluidcontained in the reservoir in a manner effective to eject a droplettherefrom.
 52. The method of claim 51, further comprising, after step(d), (d′) bombarding the designated site with photons to release samplemolecules from the sample surface for analysis.
 53. The method of claim52, further comprising, after step (d′), (e) performing massspectrometric analysis on the sample molecules.
 54. The method of claim49, wherein the sample is a cellular sample.
 55. A method for preparinga sample surface for mass spectrometry analysis, comprising: (a)providing a reservoir containing an analysis-enhancing fluid, whereinthe analysis-enhancing fluid is comprised of a mass spectrometry matrixmaterial and a carrier fluid; (b) placing a surface of a sample indroplet-receiving relationship to the fluid-containing reservoir; (c)ejecting a droplet of the analysis-enhancing fluid from the reservoirsuch that the droplet is deposited on the sample surface at a designatedsite; and (d) subjecting the sample to an atmosphere that is at leastabout 30% saturated with the carrier fluid to allow theanalysis-enhancing fluid to interact with the sample surface and torender the sample surface suitable for analysis.
 56. The method of claim55, wherein the atmosphere is at least about 50% saturated with thecarrier fluid.
 57. The method of claim 56, wherein the atmosphere is atleast about 75% saturated with the carrier fluid.
 58. The method ofclaim 57, wherein the atmosphere is at least about 90% saturated withthe carrier fluid.
 59. A method for preparing a sample surface for massspectrometry analysis, comprising: (a) providing a reservoir containingan analysis-enhancing fluid, wherein the analysis-enhancing fluid iscomprised of a mass spectrometry matrix material and a carrier fluid;(b) placing a surface of a sample in droplet-receiving relationship tothe fluid-containing reservoir; (c) ejecting a droplet of theanalysis-enhancing fluid from the reservoir such that the droplet isdeposited on the sample surface at a designated site; and (d) subjectingthe sample to conditions such that the carrier fluid exhibits avolatility quotient of at least about 30 to allow the analysis-enhancingfluid to interact with the sample surface and to render the samplesurface suitable for analysis.
 60. The method of claim 59, wherein thecarrier fluid exhibits a volatility quotient of at least about
 50. 61.The method of claim 60, wherein the carrier fluid exhibits a volatilityquotient of at least about
 75. 62. The method of claim 60, wherein thecarrier fluid exhibits a volatility quotient of at least about
 100. 63.A system for depositing an analysis-enhancing fluid on a surface of asample, comprising: a reservoir containing an analysis-enhancing fluid,wherein the analysis-enhancing fluid comprises a mass-spectrometrymatrix material and a carrier fluid, and the carrier fluid is comprisedof a low volatility solvent that has a boiling point greater than 100°C. at a pressure of 1 atmosphere; an acoustic ejector comprising anacoustic radiation generator for generating acoustic radiation and afocusing means for focusing the acoustic radiation generated; a meansfor positioning the acoustic ejector in acoustic coupling relationshipto the reservoir; and a means for positioning the sample such that adesignated site on the sample surface is positioned in droplet-receivingrelationship to the reservoir.
 64. The system of claim 63, wherein theacoustic ejector is adapted to eject a droplet having a volume nogreater than about 50 pL.
 65. The system of claim 63, further comprisinga means for applying energy to the designated site to effect release andionization of sample molecules from the sample surface for analysis. 66.The system of claim 65, wherein the energy applying means comprises ameans for bombarding the at least one selected site with photons. 67.The system of claim 66, wherein the photon bombarding means comprises alaser.
 68. The system of claim 65, further comprising an analyzerpositioned to receive ionized sample molecules released from the samplesurface.
 69. The system of claim 68, wherein the analyzer is comprisedof a mass spectrometer.